Nuclear architecture and nuclear function appear to go hand in hand, as defects in nuclear organization are associated with aging and diseases such as cancer. We have been using budding yeast as a model system to study nuclear architecture. The yeast nucleus differs from that of higher eukaryotes in two aspects: (1) yeast lack lamins, proteins that play a major structural role in shaping the nucleus in metazoans, and (2) the yeast nuclear envelope (NE) remains intact throughout the cell cycle, unlike the NE of higher eukaryotes, which breaks down during mitosis and reassembles after chromosome segregation is completed. Nonetheless, the yeast nucleus shares important features with nuclei of higher eukaryotes: the NE has to expand during the course of the cells cycle, and the nucleus maintains a spherical shape, with a volume that is proportional to cell volume. How the NE expands and what determine nuclear size and shape are questions that remain to be resolved in all systems. Our previous studies identified a number of conditions that lead to abnormal nuclear morphology, most notably elevated phospholipid synthesis (e.g. the spo7 mutant) and a cell cycle delay in mitosis. In both cases the alteration to nuclear shape is confined to the NE region adjacent to the nucleolus, and our studies showed that this confinement is dependent on the conserved polo-like kinase Cdc5. To further understand what affects nuclear morphology we sought additional proteins whose activity is required for normal nuclear size and shape. One of these turned out to be in the conserved topoisomerase 2 gene, TOP2. Cells with mutations in the TOP2 genes display multiple projection of the nuclear envelope that are not confined to the region adjacent to the nucleolus. Interestingly, only certain top2 mutants confer an abnormal nuclear shape, and this phenotype is correlated with abnormal DNA structure as determined by reduced DAPI staining. This suggests that DNA structure can affect nuclear morphology. This project is carried out in collaboration with Jeff Bachant's lab. To gain a better understanding of the relationship between nuclear and cell size, we have taken two parallel approaches: first, we asked what would happen to nuclear shape if cell growth was inhibited. To this end, we examined the consequences to nuclear morphology of inhibiting cell size expansion by inactivation the secretory pathway. Our data show that NE expansion is not linked to cell size, and that when cell growth is inhibited the nucleus deforms while maintaining the same nuclear/cell volume ratio. This observation suggests that nuclear volume, rather than its surface area, is the limiting factor for nuclear size. This work was done in collaboration with Olivier Gadal and Carolyn Larabell and was published earlier this year. Second,we began to isolate conditional mutants that have an abnormal nuclear/cell volume ratio. This work is ongoing and it is being done in collaboration with the labs of Brenda Andrews and Mike Tyers. To date we have isolated a number of mutants that affect protein accumulation in the nucleus, consistent with our conclusion from the project described above. We are currently trying to determine whether nuclear size is determined by the accumulation of a specific protein or class of proteins, or whether nuclear size is determined by the total amount of macromolecules (protein, RNA, DNA) within it.